Control of leaf scald disease

ABSTRACT

A method of substantially reducing or inhibiting the development of leaf scald disease in a plant or stalk thereof, said method comprising the step of administering an albicidin detoxification enzyme to the plant or stalk thereof. 
     There is also provided a method of generating a transgenic plant substantially resistant to albicidin and leaf scald disease including the steps of introducing and expressing a nucleotide sequence encoding albicidin detoxification enzyme into a plant, plant part or plant cell, and growing the plant, plant part or plant cell to generate the transgenic plant. 
     There is further provided a method of substantially reducing or inhibiting the development of leaf scald disease in a plant or stalk thereof, said method comprising the step of administering to the plant or stalk thereof a bacterium which extracellularly produces albicidin detoxification enzyme. 
     There is further provided an isolated albicidin detoxification enzyme capable of irreversibly inactivating albicidin as well as an isolated nucelotide sequence encoding an albicidin detoxification enzyme.

FIELD OF THE INVENTION

THIS INVENTION relates to the control of leaf scald disease and theinactivation of the phytotoxin albicidin in plants and particularly insugarcane.

BACKGROUND ART

Leaf scald is a major disease of sugarcane which occurs in more than 50countries (Chen et al., 1991, Report of the Taiwan Sugar ResearchInstitute 0 (132), 19-27; Comstock and Shine, 1992, Plant Disease 76(4), 426; Grisham et al., 1993, Plant Disease, 77 (5), 537; Irvine etal., 1993, Plant Disease, 77 (8), 846). The causal agent has beenidentified as Xanthomonas albilineans. X. albilineans produces a familyof antibiotics and phytotoxins called albicidins. Albicidins selectivelyblock DNA replication in bacteria and chloroplasts. Albicidin is thesubject of U.S. Pat. No. 4,525,354. Mutants of X. albilineans which donot produce albicidins do not produce chlorotic or any systemic diseasesymptoms in inoculated sugarcane (Birch and Patil, 1987, Physiol. Molec.Plant Pathol., 30, 199-206). This indicates that albicidins areresponsible for the chlorotic symptoms on X. albilineans infectedsugarcanes, and play an important role in sugarcane leaf scald disease.

Two different mechanisms of albicidin resistance have been identified inbacteria. One mechanism involves the loss of cell permeability in someEscherichia coli mutants to albicidin (Birch et al., 1990 J. Gen.Microbiol., 136, 51-58). The other involves the inactivation ofalbicidin by the formation of a reversible protein-albicidin bindingcomplex. This formation of a reversible binding complex has been shownin Klebsiella oxytoca to involve the albicidin resistance protein AlbA(Walker et al., 1988, Molec. Microbiol., 2 (4), 443-454) and inAlcaligenes denitrificans (Basnayake and Birch, 1995, Microbiology, 141)to involve the albicidin resistance protein AlbB. Unfortunately,however, these proteins do not irreversibly inactivate albicidin andconsequently are not considered to be efficacious candidates forcontrolling leaf scald disease.

Leaf scald disease is an economically important disease and causes alarge commercial loss in the sugarcane industry where susceptiblecultivars are grown. As a result, ways of effectively combatting thedisease are of great economic significance. For example, leaf scaldresistance in plants is an essential requirement for every commercialAustralian sugarcane variety. Selection for this resistance hasunavoidably had a significant impact on the breeding program by reducingthe value of some desired crosses and leading to the rejection of whatwould be otherwise outstanding new varieties. It takes about 10 yearsfor breeding a new sugarcane variety and rejection of one variety in thefinal stage of the breeding program would cost the industry around $1million. The recent development of a sugarcane genetic transformationsystem (Franks and Birch, 1991, Aust. J. Pit. Physiol., 18, 471-480);Bower and Birch, 1992, Plant J., 2, 409-416) has enabled the molecularimprovement of sugarcane varieties and provided a supplementarymechanism to the conventional breeding programs.

SUMMARY OF THE INVENTION

The current invention arises from the unexpected discovery of analbicidin detoxification enzyme produced from a bacterium. It wasfurther found that the albicidin detoxification enzyme was secretedextracellularly. Unlike the previously described albicidin bindingprotein AlbA and AlbB, inactivation of albicidin by the enzyme wasirreversible in the sense that albicidin toxin activity was not restoredupon protein denaturing treatment such as boiling. The bacterium thatproduced the albicidin detoxifying enzyme was identified as a strain ofErwinia herbicola also known as Pantoea dispersa.

It is therefore an object of the invention to provide an albicidindetoxification enzyme for use in treating plants infected with leafscald disease or reducing the probability of plants becoming infectedwith leaf scald disease.

It is a further object of the invention to provide a DNA sequenceencoding an albicidin detoxification enzyme for the generation oftransgenic plants and plant cells which are substantially resistant toalbicidin such that resistance to leaf scald disease is substantiallyeffected. Thus, it is yet another object to provide a transgenic plantsubstantially resistant to leaf scald disease.

Accordingly, in one aspect of the invention, there is provided analbicidin detoxification enzyme.

The term “albicidin detoxification enzyme” as used herein refers to aprotein which catalyses the conversion of an albicidin to one or morenon-toxic products wherein subsequent removal or destruction of theprotein does not result in restoration of the albicidin from thenon-toxic product(s). Accordingly, a protein being an albicidindetoxification enzyme may be distinguished from a protein whichinactivates albicidin merely by binding reversibly thereto (eg. AlbA andAlbB) by subjecting a mixture of the protein and an albicidin to aprotein denaturation step such as boiling. If the protein is analbicidin detoxification enzyme, then albicidin activity lost or reducedupon treatment with the protein is not restored by protein denaturation.Such “enzymatic detoxification” is highly advantageous because itprovides a more effective and substantially permanent protection againstalbicidin toxicity than other mechanisms mentioned heretofore which arereversed upon denaturation of a molecule which merely binds reversiblyto albicidin. It will also be appreciated that enzymatic detoxificationmay be highly beneficial in that an albicidin detoxification enzyme canprogressively detoxify multiple albicidin molecules in contrast toalbicidin binding molecules which merely bind albicidin withoutcatalytic breakdown or modification thereof.

The albicidin detoxification enzyme is preferably a hydrolase. Asuitable albicidin detoxification enzyme includes, but is not limitedto, an AlbD polypeptide comprising the sequence of amino acids as shownin FIG. 3A (SEQ ID NO:1).

Alternatively, the AlbD polypeptide is an “AlbD polypeptide homolog”.Thus, the invention contemplates polypeptides which are functionallysimilar to the AlbD polypeptide. Such polypeptides may containconservative amino acid substitutions compared to the AlbD polypeptideof FIG. 3A (SEQ ID NO:1).

The AlbD polypeptide homolog may be obtained from any suitable organismsuch as a eukaryotic cell including a yeast cell. Preferably, the AlbDpolypeptide homolog is obtained from a bacterium such as, for example,an Erwinia or Pantoea strain. Alternatively, the AlbD polypeptide orpolypeptide homolog thereof may be obtained by first isolating a DNAsequence encoding a polypeptide of the AlbD type as for exampledescribed hereinafter.

An albicidin detoxification enzyme of the invention may be prepared by aprocedure including the steps of:

(a) ligating a DNA sequence encoding an albicidin detoxification enzymeor biological fragment thereof into a suitable expression vector to forman expression construct;

(b) transfecting the expression construct into a suitable host cell;

(c) expressing the recombinant protein; and

(d) isolating the recombinant protein.

As used in this specification, an expression construct is a nucleotidesequence comprising a first nucleotide sequence encoding a polypeptide,wherein said first sequence is operably linked to regulatory nucleotidesequences (such as a promoter and a termination sequence) that willinduce expression of said first sequence. Both constitutive andinducible promoters may be useful adjuncts for expression of analbicidin detoxification enzyme according to the invention. Anexpression construct according to the invention may be a vector, such asa plasmid cloning vector. A vector according the invention may be aprokaryotic or a eukaryotic expression vector, which are well known tothose of skill in the art.

Suitable host cells for expression may be prokaryotic or eukaryotic. Onepreferred host cell for expression of a polypeptide according to theinvention is a bacterium. The bacterium used may be Escherichia coli.

The recombinant protein may be conveniently prepared by a person skilledin the art using standard protocols as for example described in Sambrooket al. (1989, Second Edition, Cold Spring Harbor Laboratory Press, 1989,in particular Sections 16 and 17).

Further, there is provided a method of substantially reducing orinhibiting the development of leaf scald disease in a plant, said methodcomprising the step of administering an albicidin detoxification enzymeto the plant.

In this case, the plant is preferably sugarcane and other plantssusceptible to leaf scald disease.

The albicidin detoxification enzyme may be combined with other agentsand may be administered by any suitable method. A suitable methodincludes soaking of stalks or setts of the plant prior to planting, andinfiltration or injection of the albicidin detoxification enzyme intothe plant.

In another aspect, the invention resides in a nucleotide sequenceencoding an albicidin detoxification enzyme. The nucleotide sequence maycomprise a nucleotide sequence encoding albD of P. dispersa.Accordingly, the nucleotide sequence may comprise the entire sequence ofnucleotides as shown in FIG. 3B (SEQ ID NO:2). Alternatively, thenucleotide sequence may comprise nucleotide 1 through nucleotide 704 ofFIG. 3B (such nucleotide sequence 1 through 704 being identified as SEQID NO:3).

The term “nucleotide sequence” as used herein designates mRNA, RNA,cRNA, cDNA or DNA.

The invention also provides homologs of the albD nucleotide sequences ofthe invention as described above. Such “albD homologs”, as used in thisspecification include all nucleotide sequences encoding sub-sequences ofthis polypeptide which confer albicidin resistance. In this regard,codon sequence redundancy means that changes can be made to a nucleotidesequence without affecting the corresponding polypeptide sequence.

The homologs of the invention further include nucleotide sequencesencoding polypeptides that have the same functional characteristics asthe AlbD polypeptides of the invention. One of skill in the art willappreciate that conservative amino acid substitutions can be made in aAlbD polypeptide according to the invention and that such substitutedpolypeptides will retain the functional characteristics of an AlbDpolypeptide according to the invention.

The homologs of the invention further comprise nucleotide sequences thathybridize with an albD nucleotide sequence of the invention understringent conditions. Suitable hybridization conditions are discussedbelow.

The albD homologs of the invention may be prepared according to thefollowing procedure:

(i) designing primers which are preferably degenerate which span atleast a fragment of a nucleotide sequence of the invention; and

(ii) using such primers to amplify, via PCR techniques, said at least afragment from a nucleic acid extract obtained from a suitable host. Inthis regard, the suitable host is preferably a bacterium such as, forexample, an Erwinia or Pantoea strain.

“Hybridization” is used here to denote the pairing of complementarynucleotide sequences to produce a DNA—DNA hybrid or a DNA-RNA hybrid.Complementary base sequences are those sequences that are related by thebase-pairing rules. In DNA, A pairs with T and C pairs with G. In RNA, Upairs with A and C pairs with G.

Typically, nucleotide sequences to be compared by means of hybridizationare analyzed using dot blotting, slot blotting, or Southern blotting.Southern blotting is used to determine the complementarity of DNAsequences. Northern blotting determines complementarity of DNA and RNAsequences. Dot and Slot blotting can be used to analyze DNA/DNA orDNA/RNA complementarity. These techniques are well known by those ofskill in the art. Typical procedures are described in CURRENT PROTOCOLSIN MOLECULAR BIOLOGY (Ausubel, et al., eds.) (John Wiley & Sons, Inc.1995) at pages 2.9.1 through 2.9.20. Briefly, for Southern blotting, DNAsamples are separated by size using gel electrophoresis. Thesize-separated DNA samples are transferred to and immobilized on amembrane (typically, nitrocellulose) and the DNA samples are probed witha radioactive, complementary nucleic acid. In dot blotting, DNA samplesare directly spotted onto a membrane (nitrocellulose or nylon). In slotblotting, the spotted DNA samples are elongated. The membrane is thenprobed with a radioactive complementary nucleic acid.

A probe is a biochemical labeled with a radioactive isotope or tagged inother ways for ease in identification. A probe is used to identify agene, a gene product or a protein. Thus a nucleotide sequence probe canbe used to identify complementary nucleotide sequences. An mRNA probewill hybridize with its corresponding DNA gene.

Typically, the following general procedure can be used to determinehybridization under stringent conditions. A nucleotide according to theinvention (such as albD or a sub-sequence thereof) will be immobilizedon a membrane using one of the above-described procedures for blotting.A sample nucleotide sequence will be labeled and used as a “probe.”Using procedures well known to those skilled in the art for blottingdescribed above, the ability of the probe to hybridize with a nucleotidesequence according to the invention can be analyzed.

One of skill in the art will recognize that various factors caninfluence the amount and detectability of the probe bound to theimmobilized DNA. The specific activity of the probe must be sufficientlyhigh to permit detection. Typically, a specific activity of at least 10⁸dpm/μg is necessary to avoid weak or undetectable hybridization signalswhen using a radioactive hybridization probe. A probe with a specificactivity of 10⁸ to 10⁹ dpm/μg can detect approximately 0.5 pg of DNA. Itis well known in the art that sufficient DNA must be immobilized on themembrane to permit detection. It is desirable to have excess immobilizedDNA and spotting 10 μg of DNA is generally an acceptable amount thatwill permit optimum detection in most circumstances. Adding an inertpolymer such as 10% (w/v) dextran sulfate (mol. wt. 500,000) or PEG 6000to the hybridization solution can also increase the sensitivity of thehybridization. Adding these polymers has been known to increase thehybridization signal. See Ausubel, supra, at p 2.10.10.

To achieve meaningful results from hybridization between a firstnucleotide sequence immobilized on a membrane and a second nucleotidesequence to be used as a hybridization probe, (1) sufficient probe mustbind to the immobilized DNA to produce a detectable signal (sensitivity)and (2) following the washing procedure, the probe must be attached onlyto those immobilized sequences with the desired degree ofcomplementarity to the probe sequence (specificity).

“Stringency,” as used in this specification, means the condition withregard to temperature, ionic strength and the presence of certainorganic solvents, under which nucleic acid hybridizations are carriedout. The higher the stringency used, the higher degree ofcomplementarity between the probe and the immobilized DNA.

“Stringent conditions” designates those conditions under which onlynucleotide sequences that have a high frequency of complementary basesequences will hybridize with each other.

Exemplary stringent conditions are (1) 0.75 M dibasic sodiumphosphate/0.5 M monobasic sodium phosphate/1 mM disodium EDTA/1%sarkosyl at about 42° C. for at least about 30 minutes, (2) 6.0Murea/0.4% sodium lauryl sulfate/0.1% SSC (20×; 3 M NaCl, 0.3 MNa₃citrate-2H₂O, pH7.0) at about 42° C. for at least about 30 minutes,(3) 0.1×SSC/0.1% SDS at about 68° C. for at least about 20 minutes, (4)1×SSC/0.1% SDS at about 55° C. for about one hour, (5) 1×SSC/0.1% SDS atabout 62° C. for about one hour, (6) 1×SSC/0.1% SDS at about 68° C. forabout one hour, (7) 0.2×SSC/0.1% SDS at about 55° C. for about one hour,(8) 0.2×SSC/0.1% SDS at about 62° C. for about one hour, and (9)0.2×SSC/0.1% SDS at about 68° C. for about one hour. See, e.g. CURRENTPROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, et al., eds.) (John Wiley &Sons, Inc. 1995), pages 2.10.1-2.10.16 of which are hereby incorporatedby reference and Sambrook, et al., MOLECULAR CLONING. A LABORATORYMANUAL (Cold Spring Harbor Press, 1989) at §§1.101-1.104.

Stringent washes are typically carried out for a total of about 20minutes to about 60 minutes. In certain instances, more than onestringent wash will be required to remove sequences that are not highlysimilar to albD or a subsequence thereof. Typically, two washes of equalduration, such as two 15 or 30 minute washes, are used. One of skill inthe art will appreciate that other longer or shorter times may beemployed for stringent washes to ensure identification of sequencessimilar to albD.

While stringent washes are typically carried out at temperatures fromabout 42° C. to about 68° C., one of skill in the art will appreciatethat other temperatures may be suitable for stringent conditions.Maximum hybridization typically occurs at about 20 to about 25° C. belowthe T_(m) for DNA—DNA hybrids. It is well known in the art that T_(m) isthe melting temperature, or temperature at which two nucleotidesequences dissociate. Methods for estimating T_(m) are well known in theart. See, e.g. Ausubel, supra, at page 2.10.8. Maximum hybridizationtypically occurs at about 10 to about 15° C. below the T_(m) for DNA-RNAhybrids.

Other typical stringent conditions are well-known in the art. One ofskill in the art will recognize that various factors can be manipulatedto optimize the specificity of the hybridization. Optimization of thestringency of the final washes can serve to ensure a high degree ofhybridization between the albD gene (or subsequence thereof) and othersimilar nucleotide sequences.

In a typical hybridization procedure, DNA is first immbolized on amembrane such as a nitrocellulose membrane or a nylon membrane.Procedures for DNA immobilization on such membranes are well known inthe art. See, e.g., Ausubel, supra at pages 2.9.1-2.9.20. The membraneis prehybridized at 42° C. for 30-60 minutes in 0.75 M dibasic sodiumphosphate/0.5 M monobasic sodium phosphate/1 mM disodium EDTA/1%sarkosyl. Membranes are then hybridized at 42° C. in ACES hybridizationsolution (Life Technologies, Inc., Gaithersburg, Md.) containing labeledprobe for one hour. Next, membranes are subjected to two high stringency10 minute washes at 42° C. in 0.75 M dibasic sodium phosphate/0.5 Mmonobasic sodium phosphate/1 mM disodium EDTA/1 % sarkosyl. Followingthis, the membranes are washed with 2×SSC. at room temperature, toremove unbound probe.

In another typical hybridization procedure, DNA immobilized on amembrane is hybridized overnight at 42° C. in prehybridization solution.Following hybridization, blots are washed with two stringent washes,such as 6.0M urea/0.4% sodium lauryl sulfate/0.1% SSC. at 42° C.Following this, the membranes are washed with 2×SSC. at roomtemperature.

Autoradiographic techniques for detecting radioactively labeled probesbound to membranes are well known in the art.

There is also provided a method of generating a transgenic plantsubstantially resistant to albicidin and leaf scald disease, said methodincluding the steps of introducing and expressing a nucleotide sequenceencoding albicidin detoxification enzyme into a plant, plant part orplant cell, and growing the plant, plant part or plant cell to generatethe transgenic plant.

The invention also comprises a method of generating a transgenic plantsubstantially resistant to albicidin and leaf scald disease, said methodincluding the steps of introducing into a plant, or plant part or cellthereof a vector comprising a nucleotide sequence encoding an albicidindetoxification enzyme wherein said nucleotide sequence is operablylinked to one or more regulatory nucleotide sequences and growing saidplant or plant part or cell thereof to generate the transgenic plant.

The nucleotide sequence encoding the albicidin detoxification enzyme mayinclude any of the sequences described above. The nucleotide sequencemay comprise the entire sequence of nucleotides as shown in FIGS. 3B-3F(SEQ ID NO:2). Preferably, the nucleotide sequence comprises nucleotide1 through nucleotide 704 of FIGS. 3B-3F (SEQ ID NO:3).

The plant, plant part or plant cell may be obtained from any suitableplant which could be infected with leaf scald disease. Preferably, theplant, plant part or plant cell is obtained from a sugarcane variety.

In another aspect, the invention provides a transgenic plantsubstantially resistant to albicidin and leaf scald disease, said plantcomprising a nucleotide sequence encoding an albicidin detoxificationenzyme wherein said sequence is operably linked to one or moreregulatory nucleotide sequences.

Preferably, the nucleotide sequence is stably incorporated within cellsof said plant.

Of course, it will be appreciated that if the albicidin detoxificationenzyme requires transportation to a specific cellular compartment inorder to effect resistance to albicidin, such transportation may beeffected by construction of a translational fusion comprising thealbicidin detoxification enzyme fused in frame with a DNA sequenceencoding a transit peptide. Such transit peptides are well known in theart and may include, for example, a plastid transit peptide such as themaize waxy transit peptide as for example described in an article byKlösgen and Weil (1991, Molec. Gen. Genet., 225, 297-304) which ishereby incorporated by reference. This transit peptide has been used intargeting a range of proteins to the plastids of a range of plantspecies, for example in locating the NPT II protein to tobaccochloroplasts (Van den Broeck et al., 1985, ) and in locating GUS proteininto chloroplasts of potato plants (Klösgen and Weil, 1991, Nature, 313,358-363).

A vector according to the invention may be a prokaryotic or a eukaryoticexpression vector, which are well known to those of skill in the art.Such vectors may contain one or more copies of the nucleotide sequencesaccording to the invention.

Regulatory nucleotide sequences which may be utilized to regulateexpression of the nucleotide sequence encoding the albicidindetoxification enzyme include, but are not limited to, a promoter, anenhancer, and a transcriptional terminator. Such regulatory sequencesare well known to those of skill in the art.

Suitable promoters which may be utilized to induce expression of thenucleotide sequences of the invention include constitutive promoters andinducible promoters. A particularly preferred promoter which may be usedto induce such expression includes the p_(EMU) monocots promoter asdescribed for example in U.S. Pat. No. 5,290,924 (Last et al), and theplant ubiquitin promoter p_(UBI) as described for example in EP342926(Quail).

Any suitable transcriptional terminator may be used which effectstermination of transcription of a nucleotide sequence in accordance withthe invention. Preferably, the nopaline synthase (NOS) terminator, asfor example disclosed in U.S. Pat. No. 5,034,322, is used as thetranscription terminator.

The vector may also include a selection marker such as an antibioticresistance gene which can be used for selection of suitabletransformants. Examples of such resistance genes include the nptil genewhich confers resistance to the antibiotics kanamycin and G418(Geneticin®) and the hph gene which confers resistance to the antibiotichygromycin B.

A nucleotide sequence or vector according to the invention may beintroduced into a plant, or plant part, or cell thereof using anysuitable method including transfection, projectile bombardment,electroporation or infection by Agrobacterium tumefaciens.

It will of course be appreciated that gene transplacement by homologousrecombination may also be used to effect the generation of suitabletransgenic plants. Such methods are well known to persons of skill inthe art.

In yet another aspect of the invention, there is provided a bacteriumwhich can produce an albicidin detoxification enzyme for use in treatingplants infected with leaf scald disease and/or reducing the probabilityof plants becoming infected with leaf scald disease.

The bacterium may be any suitable strain derived from a naturallyoccurring strain capable of producing albicidin detoxification enzymewhen selected by procedures outlined in the preferred embodiment. Asuitable bacterium may be a strain of Erwinia herbicola such as E.herbicola SB1403 (also known as Pantoea dispersa SB1403). A descriptionof E. herbicola SB1403 is given in the preferred embodiment. This strainhas been deposited with the Australian Government AnalyticalLaboratories on Apr. 11, 1995 with the accession number N95/21834.

Alternatively, the organism may be any suitable strain capable ofexpressing extracellularly a nucleotide sequence encoding the albicidindetoxification enzyme as herein described. Suitable strains include E.coli and suitable soil or plant commensal bacteria harbouring a copy ofthe gene encoding albicidin detoxification enzyme.

There is also provided a method of substantially reducing or inhibitingthe development of leaf scald disease in a plant or stalk thereof, saidmethod comprising the step of administering to the plant or stalkthereof a bacterium which extracellularly produces albicidindetoxification enzyme. The method may include as the biocontrol agent astrain of P. dispersa or a suitable host expressing a cloned sequenceencoding albicidin detoxification enzyme.

The strain may be administered by any suitable method including sprayingon the foliage. Other examples or administration include the dripping ofcultures onto base cutters or cutter-planters, or through spray nozzlesdirected at freshly cut stubble. The biocontrol agent may be combinedwith one or more other agents which facilitate its operation or performadditional tasks. Other agents may include fungicides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 refers to a time course of albicidin detoxification by cell freeextracts of E. herbicola SB1403;

FIG. 2 illustrates a physical map of QZB103 and derivatives thereof;

FIG. 3A designates the predicted polypeptide sequence encoded by thealbD gene (SEQ ID NO:1);

FIGS. 3B-3F show the nucleotide sequence of the albD gene (SEQ ID NO:2);

FIG. 4 refers to a best matched comparison of amino acid sequencesencoded by the albD gene product (SEQ ID NO:4) and the proteinsrespectively encoded by the A. denitrificans albB gene (SEQ ID NO:5) andby the K. oxytoka albA gene (SEQ ID NO:6);

FIG. 5A illustrates the internal HincII-StuI fragment of the albD gene;

FIG. 5B refers to a map of plasmid pJPAldHS;

FIG. 6 refers to a graph showing inactivation of albicidin by E. coliDH5α [pQZE533] and E. coli DH5α [pSB6];

FIG. 7A illustrates a 790 base pair albD structural gene fragment whichwas amplified using PCR and specific flanking oligonucleotide primersSEQ ID NO:7 and SEQ ID NO:8, respectively;

FIG. 7B illustrates the position of the thrombin cleavage site upstreamof the predicted initiation codon of albD (peptide identified as SEQ IDNO:9; nucleotide base identified as SEQ ID NO:10);

FIG. 7C refers to a map of GST-albD gene fusion construct pGSTALD;

FIG. 8 shows a bar graph of the effect of temperature on AlbD enzymeactivity;

FIG. 9 represents a bar graph illustrating the effect of pH on AlbDenzyme activity;

FIG. 10A (SEQ ID NO:7 and SEQ ID NO:8) is the same as FIG. 7A;

FIG. 10B refers to a map of a plasmid clone designated pTacAld showingthe orientation of the albD structural gene portion under the influenceof the tac promoter;

FIG. 11A illustrates a map of the sugarcane expression vector pU3Z;

FIG. 11B (SEQ ID NO:7 and SEQ ID NO:8) is the same figure as FIG. 7A;

FIG. 11C. refers to a map of construct pU3ZALD;

FIG. 12 refers to a bar graph showing the frequency distribution ofdisease severity in sugarcane cultivar Q63 plant lines regenerated fromcallus co-bombarded with albD wherein the plant lines were inoculatedwith X. albilineans XA3; and

FIG. 13 illustrates a bar graph showing the frequency distribution ofdisease severity in sugarcane cultivar Q63 plant lines regenerated fromcallus co-bombarded with albD wherein the plant lines were inoculatedwith X. albilineans XA15.

PREFERRED EMBODIMENTS EXAMPLE 1 Expression of Albicidin DetoxificationEnzyme from Pantoea dispersa in Transgenic Sugarcane

MATERIALS AND METHODS

Bacteria and cultivation. The bacterial strains and plasmids used inthis work are listed in TABLE 1. E. coli strain DH5α was used asalbicidin activity indicator strain and also as the host strain for DNAcloning and subcloning; X. albilineans strain XA3 isolated from diseasedsugarcane from Queensland, Australia was used as albicidin productionstrain; the different albicidin-resistant (Alb^(r)) isolates isolatedfrom X. albilineans infected sugarcanes were listed in TABLE 2. E. colistrains were grown and maintained in LM medium (Miller, 1972,Experiments in Molecular Genetics, Cold Spring Harbour, N.Y.: ColdSpring Harbor Laboratory Press), the others in SP medium (Birch andPatil, 1985, J. Gen. Microbiol., 131, 1069-1075). Except for E. colistrains that were usually grown at 37° C., all the other strains andisolates were grown in 28° C. Broth cultures aerated by shaking at 200rpm on an orbital shaker.

Isolation of bacteria. X. albilineans infected sugarcane samples werecollected from Eight Mile Plains, Queensland, Australia. These sampleswere surface sterilised by 70% ethanol and then segmented into smallpieces. The pieced samples were suspended in sterilised water and shakenin a reciprocal shaker for 1 h before spreading over SP agar plates(Birch and Patil, 1985, J. Gen. Microbiol., 131, 1069-1075). Thecolonies which appeared were collected based on morphology for furtheranalysis.

Preparation of albicidin. Albicidins produced in culture by X.albilineans were purified as described previously (Birch and Patil,1985, J. Gen. Microbiol., 131, 1069-1075; and Birch et al., 1990, J.Gen. Microbiol., 136, 51-58). Unless stated otherwise, the mixture ofalbicidins obtained after HW-40(s) chromatography was used inexperiments reported here.

Albicidin bioassay. Albicidin was quantified as described previously(Birch and Patil, 1985, J. Gen. Microbiol., 131, 1069-1075).

Inactivation of albicidin by intact bacterial cells. Actively growingbacterial culture with OD₆₀₀ equal to about 1.5 was added to equalvolume of SP or LM liquid broth containing albicidin with a finalconcentration of 1000 units/mL. Samples were removed at intervals andplaced on ice or boiled for 5 min. After reaction, samples werecentrifuged and supernatants were collected. For unboiled treatment,supernatants were exposure to UV light for 10 min before bioassay.

Cell free extract preparation. Bacterial isolates were inoculated in 100ml of SP liquid medium and grown for 24 h with shaking at 28° C. Cellswere harvested by centrifugation at 11020×g for 10 min and washed inTEMM buffer (10 mM Tris pH 7.45, 10 mM EDTA, 10 mM MgCl₂ and 2 mMβ-mercaptoethanol). The cells were resuspended in 2 ml TEMM buffer anddisrupted by sonification on ice with a mircroprobe (model 250, BransonUltrasonic Corporation, Danbury, Conn.) at 50% duty cycle and an outputsetting of 3. Sonification was performed for 3 min of 8 second burstperiods followed by 8 second rest periods. Cell disruption was confirmedby using phase contrast microscopy. Cell debris was removed bycentrifugation at 11020×g for 20 min. The protein concentration in thecell extracts was then determined by dye reagent method (Bradford, 1976,Analytical Biochemistry, 72, 248-254) using bovine serum albumin asstandard control.

Taxonomic identification method. Gram reaction was tested by using KOHmethod (Suslow et al., 1982, Phytopathology, 72, 917-918). Utilisationof different carbon sources were tested by using BIOLOG GN Microplate™(Biolog Inc.). Oxidation fermentation assay was performed by using Hughand Leifson (HL) test (Collins and Lyne, 1984, Microbiological Methods,5th Ed. London, Butterworths).

Plant material and bacterial inoculation. Sugarcane variety Q44 was usedin all biocontrol experiments, single-node cuttings from X. albilineansfree healthy sugarcane plants were potted in PH1 contaminationgreenhouse for about two months before inoculation. A decapitationmethod described previously (Birch and Patil, 1983, Phytopathology, 73,1368-1374) was used for inoculation of X. albilineans and E. herbicolastrains. The actively growing bacteria (2 days culture of X. albilineansstrains and 24 h culture of E. herbicola strains) were centrifuged for 1min at 14000 rpm, resuspended and diluted with sterilised water torelevant concentrations, and kept on ice until inoculation.

Cloning and sequencing of the albD gene. A cosmid genomic library of E.herbicola SB1403 was constructed in E. coli DH5α by partial digestion ofthe SB1403 DNA with BamHI restriction endonuclease and ligation into theBamHl site of the cosmid cloning vector pLAFR3. Albicidin resistantrecombinant clones were selected by patching the recombinant cosmidclone transformants onto LB agar medium containing 10 μg/mL Tc and 20u/mL albicidin. T6 phage sensitivity was confirmed by cross streaking.Subcloning into pBluescript II SK(+) was carried out according toroutine techniques (Sambrook et al., 1989. NY: Molecular Cloning: ALaboratory Manual, 2nd Ed. Cold Spring Harbour Laboratory.).

Alb^(r) clone pQZE533 and four ExoIII deletion sub-clones (pQZE456,pQZE457, pQZE540 and pQZE560) were used to obtain the complete DNAsequence from both strands of the cloned albD gene. DNA sequencing wasbased on the dideoxynucleotide chain termination method of (Sanger etal., 1977, PNAS USA, 74, 5463-5467). The PRISM™ Ready Reaction DyeDeoXy™Terminator Cycle Sequencing Kit was from Applied Biosystems.

Sited directed mutagenesis of albD gene in E. herbicola SB1403. A 326 bpHincII-StuI fragment, constituting an internal segment of the albD geneof E. herbicola SB1403 was ligated into the suicide vector pJP5603. Theligation products were used to transform E. coli JM109 (λpir). Arecombinant clone pJPAldHS was identified by restriction enzymedigestion agarose gel electrophoresis. It was transferred into themobilising strain S17-1 (λpir), and mobilised into E. herbicolaSB1403rif. Exconjugant colonies were selected on SP agar mediumcontaining 50 μg/mL kanamycin and 50 μg/mL rifampicin, and tested foralbicidin detoxification enzyme production.

PCR amplification and modification of albD gene. A plasmid clone pQZE533containing albD gene was used as a template for PCR amplification. Twooligonucleotide primers were synthesised corresponding to the 5′ and 3′flanking regions of albD structural gene. The 5′ and 3′ primers were(5′-TTMG CGGGA TCCGT TTTGA TGGAC-3′) (SEQ ID NO:7) and (5′-GATTG MTCGTATCA GCTGG MGAG-3′) (SEQ ID NO:8), respectively.

The PCR reaction was performed in a reaction volume of 100 μL using 2 ngof pQZE533 DNA, primer concentrations of 0.4 ng/μL, 400 μM of each ofthe deoxynucleoside triphosphates, 2 mM MgCl₂, 0.5 units of Vent (exo⁻)DNA polymerase and 1×PCR reaction buffer (New England Biolabs) aPerkin-Elmer Cetus DNA thermal cycler machine was used for the reactionat an initial heat denaturation temperature of 95° C. for 5 min, then 30cycles at a denaturation temperature of 95° C. for 1 min, an annealingtemperature of 55° C. for 1 min and a polymerisation temperature of 72°C. for 1 min. A final polymerisation temperature of 72° C. for 7 min wasused following completion of the 30 cycles. An aliquot (10 μL) from eachof the completed PCR reactions was subjected to electrophoresis in a 1%agarose gel and the products visualised following ethidium bromidestaining and UV transillumination. The PCR product of 790 bp waspurified by phenol-chloroform extraction and ethanol precipitation. Thepurified PCR product was dissolved in LTE buffer (10 mM Trizma Base, 1mM Na₂EDTA, pH8.0) and kept in −20° C. before use.

Purification of AlbD Enzyme.

The PCR amplified structural gene fragment was digested by BamHI andPvuII and ligated to the BamHI and SmaI digested GST gene fusion vectorpGEX-2T. The resultant construction pGSTAld contains the chimeric albDgene fused in frame to the glutathione S-transferase (GST) gene which isunder the control of IPTG inducible tac promoter (FIG. 10).

E. coli DH5α (pGSTAld) was cultured and induced by IPTG. Thepurification of AlbD enzyme was basically following manufacturer'sinstruction (Pharmacia). Briefly, the bacterial culture was pelleted bycentrifugation and cell free extracts were prepared by ultrasonificationand applied to the Glutathione Sepharose 4B affinity column. TheGST-AlbD fusion protein was bound to the affinity column matrix, and theAlbD enzyme protein was separated from the GST protein by digestion withprotease Thrombin for 16 hours at room temperature. Following digestion,the eluate containing pure AlbD protein was collected and analysed bySDS-PAGE. The purified enzyme was kept in −20° C. in PBS buffer (140 mMNaCl, 2.7 mM KC1, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH7.3) plus 20%glycerol.

Monocot expression vector construction. The basis of the monocotexpression vector constructs was pGEM-4Z. A 260 bp SstI-EcoRI fragmentcontaining the terminator sequence (nos3′) from the nopaline synthasegene of the Agrobacterium Ti plasmid was isolated from GUS gene fusionplasmid pBI101 (CLONTECH) and inserted into the SacI-EcoRI site ofpGEM-4Z. The HindIII-BamHI fragment about 1.9 kb in size containingubiquitin promoter and intron sequences was isolated from the constructpAHC18, which contains ubiquitin-promoter/luciferase (ubi-luc) fusiongene (Bruce et al., 1989. Proc. Natl. Acad. Sci. USA., 86, 9692-9696).This fragment was then ligated to the HindIII-BamHI sites of thepGEM-4Z::nos 3′. The resultant monocot expression vector pU3Z hasseveral unique restriction enzyme sites such as BamHI, SmaI, KpnI andSacI in between promoter/intron and terminator region for subsequentcloning genes of interest (FIG. 11A).

pU3Zald and pU3ZGUS construction and transformation of sugarcane. Toconstruct pU3Zald (FIG. 11C), the ubi-albD fusion gene, the PCRamplified BamHI-PvuII fragment of albD structural gene (FIG. 11B) wasligated to the pU3Z (FIG. 11A) linearized by BamHl and Smal. The pU3ZGUSwas constructed by fusion of the BamHI-SstI GUS fragment from pBI101(CLONTECH) to the BamHI and Sacl sites of vector pU3Z (map not shown).Transformation of sugarcane is briefly outlined as follows.

Embryogenic callus of the sugarcane cultivar Q63 was established andmaintained as described by Franks and Birch, (1991), Aust. J. Plt.Physiol., 18, 471-480. The embryogenic callus was placed in a circle of2.5 diameter on an osmoticum plate (MSC₃ plus 0.2M mannitol an 0.2Msorbitol) at 4 h prior to bombardment. The callus was bombarded withDNA-coated tungsten microprojectiles using the apparatus and techniquesdescribed by Franks and Birch (1991), Aust. J. Plt. Physiol., 18,471-480. After bombardment the callus was kept on the same osmoticumplates for another 4 h before being transferred to MSC3 (Heinz and Mee,1969) selective medium.

Selection procedures and regeneration of transgenic sugarcane. Followingbombardment with constructs described under the sub-heading “Productionand analysis of transgenic plants” as described hereinafter, embryogeniccallus was cultured on an initial selection medium containing 20 μg/mLgeneticin for 2 weeks. Healthy callus was transferred to mediumcontaining 30 μg/mL geneticin for another 2 weeks. Escape-free selectionwas then applied by transferring the healthy callus to a MSC₃ mediumcontaining 45 μg/mL geneticin for about 4 weeks. Then actively growingcallus was transferred onto a regeneration MSC medium containing thesame concentration of antibiotics. These regeneration plates were placedin the tissue culture room at 28° C. under fluorescent lighting. Afterabout 2 weeks culture, small plantlets were separated and placed on thesame regeneration medium for further growing until ready forestablishment in pots.

Detection of AIbD enzyme expressed in transgenic sugarcane. One gram ofsugarcane leaves were cut into small pieces and frozen in liquidnitrogen, ground into powder in a mortar before adding 4 ml ofextraction buffer (100 mM KPO₄, 10 mM DTT, 1 mM EDTA, 3% Triton X-100,pH 7.0) buffer for further grinding for 1 min; transferred into a 10 mLtube and allowed to stand 30 mins in an ice box. Supernatants werecollected after centrifugation at 4° C. for 20 min (14000 rpm). Proteinconcentrations in the supernatants were measured. Supernatants wereadded to albicidin solution and incubated for 2 h at 28° C. beforebioassay. Disappearance of albicidin from the reaction mixture indicatedthe presence of AlbD enzyme.

RESULTS AND DISCUSSION

Isolation of Albicidin Resistant Bacteria

Fifteen different bacterial isolates that show differences in size,colour and shape of colonies on non-selective medium were collected fromdifferent parts of the X. albilineans infected sugarcane. Among them,thirteen isolates showed different levels of albicidin resistance, someare highly resistant (1000 u albicidin/mL), whereas others only showmoderate or low levels of resistance (TABLE 2). In this investigation,one activity unit of albicidin was defined as the amount of toxin thatcould produce a 3 mm inhibition zone in the plate overlay bioassay(Birch and Patil, 1985, J. Gen. Microbiol., 131, 1069-1075).

Screening of Bacteria that can Produce Albicidin Detoxification Enzyme

Albicidin is heat stable and its activity is unaffected at 100° C. for30 min (Walker et at., 1988, Molec. Microbiol., 2 (4), 443-454), whereasbacterial cell membrane and many proteins can be denatured by boilingfor a short time. A simple and efficient assay was therefore designed todetect different albicidin resistant mechanisms. Bacterial isolatesresistant to 500 u/mL albicidin were tested for their possibleresistance mechanism. Some bacterial strains of known albicidinresistance mechanism were used as controls. The fresh bacterial cultureswere mixed with albicidin solution, and the mixture was divided into twoparts after incubation. One part was boiled, the other remain unboiled.Then the supernatants were assayed for albicidin. The results in TABLE 3shows that albicidin activity was recovered by boiling the cells of A.denitrificans and E. coli (pBS6) which is the typical of reversibletoxin binding mechanism (Basnayake and Birch, 1995, Microbiology, 141;Walker et al., 1988, Molec. Microbiol., 2 (4), 443-454). E. coli strainRR1 Alb^(r), excludes albicidin from entering the bacterial cells. Bycomparison with these controls, we can classify those albicidinresistant isolates into three groups with different putative resistantmechanisms. Isolates SB1401 and SB1402 are likely to have a toxinreversible binding mechanism like A. denitrificans and E. coli (pBS6).The resistant mechanism of SB501, SB1301 and SB1404 could be toxinexclusion or resistant target. Isolates SB101, SB107 and SB1403 thatwere able to detoxify albicidin irreversibly are most likely to havealbicidin detoxification enzymes. Among them, strain SB1403 shows thestrongest albicidin detoxification activity.

It is confirmed by the cell free extracts experiment that strain SB1403can produce an albicidin detoxification enzyme. FIG. 1 is the timecourse reaction of cell free extracts of SB1403. In the presence ofactive cell free extracts, albicidin has been rapidly and progressivelyremoved from the reaction mixture. But almost all toxin added remainedin the reaction mixture if the cell free extracts were denatured byboiling before reacting with albicidin.

Identification of Strain SB1403

Strain SB1403 is gram-negative, ONPG test positive, motile androd-shaped bacterium with 4-8 peritrichous flagella. Its cell size wasabout 0.6-1.0 μm wide×1.3-3.0 μm long. It can produce yellow pigment onSP medium. A positive reaction resulted in the test for oxidation orfermentation of glucose in Hugh and Leifson's medium. The strain wasfurther classified by using GN MicroPlates which contain 95 carbonsource utilisation tests (BIOLOG). In this assay, utilisation of acarbon is detected as an increase in the respiration of cells in thewell, leading to irreversible reduction of tetrazolium dye. The“breathprint” thus obtained was matched to the Gram-negative Databasecontaining identification patterns of 569 Gram-negative species/groups.The matching results show that the isolate SB1403 was Erwina herbicola(Enterobacter agglomerans A).

Biocontrol of Leaf Scald by E. herbicola SB1403

TABLE 4 shows that E. herbicola SB1403 was very effective in thebiocontrol of leaf scald disease. X. albilineans caused a severe damageto sugarcane Q44, 50% of the newly emerged leaves after inoculation weredead, and there were 139 white pencil lines observed in survivingleaves. But in those plants co-inoculated with E. herbicola SB1403, noneof the leaves was dead and only 2 or 3 white pencil lines were observed.Furthermore, biocontrol agent E. herbicola did not have any detectableside effect on sugarcane.

Some antibiotic producing E. herbicola isolates are used in biocontrolof fire blight, a disease of rosaceous plants caused by Erwiniaamylovora (Vanneste et al., 1992, Journal of Bacteriology, 174,2785-2796). But E. herbicola SB1403 did not produce any detectableantibiotic against E. coli and X. albilineans. Determination of the roleof albicidin detoxification enzyme production by E. herbicola SB1403 inthe biocontrol of leaf scald disease will be described in a latersection.

Cloning Albicidin Detoxification Gene

About 1600 Tc^(r) cosmid clones were patched on to LB plates containing500 u/mL albicidin, and 5 albicidin resistant strains were detected. Thecosmids from 4 strains were digested with BamHl and all found to containa 8 kb common band. This common fragment was cloned into the DNAsequencing vector pBluescript and named pQZB103 (FIG. 2). The plasmidDNA of pQZB103 was partially digested by HincII and religated. Thereligation products were used to transform E. coli DH5α andtransformants were selected on LB plates containing albicidin. Theplasmids of albicidin resistant colonies were isolated and their sizeswere determined by agarose gel electrophoresis. Restriction enzymedigestion of the smallest plasmid clone pQZH301 showed that it containedonly two HincII fragments, one is 1.9 kb in size and the other is 0.6kb. A range of subclones of pQZB301 were obtained by double exonucleaseIII unidirectional deletions and religation to the cloning vector. Eachof these plasmids was transformed into E. coli DH5α and sensitivity ofeach to albicidin was determined (FIG. 2). All of the Alb^(r) clonesremained sensitive to bacteriophage T6 infection, indicating resistanceis not due to the spontaneous mutation of the Tsx pore involved inalbicidin uptake (Birch et al., 1990, J. Gen. Microbiol, 136, 51-58).

Nucleotide Sequence of the Albicidin Detoxification Gene

The DNA sequence and the inferred amino acid sequence of a portion ofthis albD gene is shown on FIGS. 3A and FIGS. 3B-3F. We find only oneopen reading frame reading, which could encode a hydrophilic protein of235 amino acids, having a molecular weight of 24511 daltons. Of the 235amino acids in the AlbD protein, there are 59 charged residues; 32 areacidic and 27 are basic, resulting an isoelectric point at 6.23. Thebest complementary sequence to the 16s rRNA 3′-UCUUUCCUCCACUA sequence(SEQ ID NO:11) was found 10 bp upstream from the ATG initiation codonleading the only open reading frame (shadowed), but it does not matchwell to the AGGAGG Shine-Dalgarno sequence (Shine and Dalgarno, 1974,Proc. Natl. Acad. Sci. USA., 71, 1342-1346).

The transcription termination site of albD possibly belongs to thefactor-independent group (Platt, 1986, Annu. Rev. Biochem., 55,339-372). Two TCTT boxes and a TGTG box that are closely resemble theTCTG consensus sequence characteristic of factor-independent terminationsites (Brendel and Trifonov, 1984, Nucleic Acids Research, 12,4411-4427) were found downstream of the termination codon of albD gene.Besides, T-rich regions are located upstream of the two TCTT boxesalthough T-content is not highly significant. But there is no GC-richdyad symmetry region downstream the termination codon.

The FASTA program of Lipman and Pearson was used to compare the DNA(FIGS. 3B-3F) (SEQ ID NO:2) and protein sequences (FIG. 3A) (SEQ IDNO:1) of this gene to all DNA and Protein sequences in major sequencedatabases (GenBank, EMBL, PIR and Swiss-Prot) through the AustralianNational Genomic information Service. However, no significant similarityhas been detected to any known DNA or protein sequences. In this regard,the prior art sequences exhibiting the greatest homology at the proteinlevel comprised mouse T-cell-specific transcription factor −1P (28.7%identity in a 136 aa overlap, PIR Accession #JH0401); Agrobacteriumtumefaciens hypothetical protein 2 (29.0% identity in a 107 aa overlap,PIR Accession #S07977); Bacillus subtilis dihydroorotase (30.5% identityin a 95 aa overlap, PIR Accession #D39845); Rhizobium meliloti flagellinflaA (21.4% identity in a 154 aa overlap, PIR Accession #A39436);Pseudomonas cepacia beta-lactamase (22.4% identity in a 210 aa overlap,PIR Accession #A48903); and Xanthomonas campestris copD homolog (28.3%identity in a 152 aa overlap, PIR Accession #D36868).

We also compared the AlbD protein sequence with the conserved region ofthe two known albicidin binding proteins (Walker et al., 1988, Molec.Microbiol., 2 (4), 443-454; Basnayake and Birch, 1995, Microbiology,141). FIG. 4 shows the best match of the AlbD amino sequence to thefirst 16 amino acids at the N terminus of the Alb^(r) binding proteinsfrom K. oxytoca and A. denitrificans respectively. This shortoligopeptide is the only significantly conserved region in the twoproteins and is likely to be the albicidin binding domain (Basnayake andBirch, 1995, Microbiology, 141). As shown in FIG. 4, the motif for thesethree albicidin resistant proteins seems to be “SxxxLxxL” or lessstrictly “MYxxxFSxxxLxxLxL” (SEQ ID NO:12).

The Role of AlbD Enzyme of E. herbicola SB1403 in Biocontrol of X.albilineans

E. herbicola SB1403 provided a very effective biocontrol of leaf scalddisease caused by X. albilineans (TABLE 4). To establish the role ofalbicidin detoxification enzyme produced by SB1403 in the control ofleaf scald, we isolated site-directed mutants of SB1403 that lost theability to produce AlbD enzyme, and compared their effectiveness in thebiocontrol of leaf scald to that of their parent strain.

A “universal” suicide vector pJP5603 that replicates only if the R6K pirgene is supplied in trans was used for generation of albD gene insertionmutation in SB1403 (Penfold and Pemberton, 1992, Gene, 118, 145-146). AHincII-StuI internal fragment of AlbD gene was cloned into pJP5603, theresultant recombinant clone pJPAldHS (FIG. 5) was mobilised into SB1403.Transconjugant colonies were obtained at a frequency of 3.5×10⁻⁷, and75% of colonies lost their ability to produce AlbD enzyme, indicatingthat albD has been successfully mutated. This also shows that the albDgene is not essential for bacterial growth and that it is a single copygene.

Two AlbD⁻ mutants of SB1403 have been tested in biocontrol of leaf scalddisease. TABLE 5 shows that sugarcane co-inoculated with X. albilineansXA3 and E. herbicola SM1 or SM18 had about 5 times more white pencillines compared to those plants treated with XA3 and SB1403rif. Thesedata indicate that AlbD enzyme is contributing to biocontrol of leafscald.

Comparison of the Activities of Cloned albB and albD Gene Products in E.coli

FIG. 6 shows the relative albicidin inactivation activities of E.coliDH5α [pSB6] containing albB cloned from A. denitrificans and E. coliDH5α [pQZE533] containing albD gene cloned from E. herbicola SB1403.DH5α [pQZE533] gradually removed albicidin from the reaction mixture andgave 100% irreversible detoxification of albicidin within 120 min.Strain DH5α [pSB6] reduced anti-microbial activity by more than 50%within the first 15 min, but there was no further reduction. This ispossible because binding proteins bind and thus inactivates albicidin ina molar ratio of 1 binding protein: 1 albicidin (Basnayake and Birch,1995, Microbiology, 141). Protein bound albicidin is inactive in theanti-microbial assay. Once the binding protein pool was exhausted byforming unrecyclable protein:albicidin complexes, DNA synthesis in thebacteria cell could be inhibited by excessive albicidin. Reducedanti-microbial activity of albicidin by binding protein is reversible; alarge proportion of toxin activity was released when theprotein:albicidin complex was denatured by boiling. The data indicatethat AlbD enzyme results in irreversible detoxification of albicidinthan AlbB binding protein, a much more effective method of albicidinresistance than reversible interaction with albicidin binding protein.

Purification and Properties of AlbD Enzyme

GST gene fusion system (Smith and Johnson, 1988. Gene 67: 31-40) wasused to purify the albicidin detoxification enzymes, the product of albDgene. The PCR amplified albD structural gene portion was fused to the C.terminus of glutathione S-transferase (GST) gene in the same openreading frame (FIG. 7). The fusion protein expressed in E. coli DH5αafter induction by IPTG was bound to the GST affinity column. The pureAlbD enzyme protein was released from the column after digestion of thefusion protein with site specific protease Thrombin which recognises thecleavage-recognition sequences at the recombinant C terminus of the GST.SDS/PAGE analysis showed that the purified AlbD enzyme has a molecularmasses of about 25.5 kDa, this is consistent with the predicated 25411kDa molecular masses of the AlbD protein.

The purified AlbD enzyme can be stably maintained in 20% glycerol bufferat −20° C. FIG. 8 shows that the enzyme can detoxify albicidin at 45°C., but prefers mild temperatures with the maximum activity at 28° C.FIG. 9 shows that the AlbD enzyme was not sensitive to changes of pH inthe reaction solutions; the enzyme detoxified albicidin almost equallywell in a range from pH 5.8 to pH 8.0.

As purified enzyme can effectively detoxify relatively pure albicidin ina very simple phosphate buffer, it seems that the AlbD enzyme does notrequire any complex cofactor for its activity.

These properties of AlbD enzyme suggest that it would work efficientlyin the cytoplasm or plastids of plant cells.

DNA modification for Expression in Plant

In the native albD gene, there is an out of frame ATG initiation codon 8bp upstream of +1 bp which could interfere with the high levelexpression of the albD gene when transferred into plants. This spuriousstart codon was eliminated by incorporating a point mutation in the PCRforward primer of the albD gene. As a result, the ATG has been changedto ATC, and this change also creates a restriction enzyme BamHI site onthe 5′ end of the albD gene PCR product which was used in the subsequentcloning (FIG. 10).

The PCR amplified promoterless albD structural gene portion was fused tothe tac promoter of the bacterial expression vector pKK223-3(Pharmacia). One of the resultant clones, pTacAld was identifiedcontaining correctly oriented tac-albD fusion gene (FIG. 10). Thecorrect function was confirmed by demonstration of albicidindetoxification by pTacAld transformed E. coli (data not shown).

Production and Analysis of Transgenic Plants

To test whether this novel albicidin detoxification gene can also conferresistance to leaf scald disease in sugarcane, the PCR amplifiedpromoterless albD structural gene portion was inserted in between theubi-intron promoter region and the nopaline synthase polyadenylationsignal on the monocot expression vector pU3Z (FIG. 11). The resultingplasmid pU3ZAld was used for transformation of the chimeric albD geneinto sugarcane (Q63) by microprojectile bombardment (Bower and Birch,1992, Plant J., 2, 409-416). The neomycin phosphotransferase (npt-II)gene encoding resistance to geneticin under the control of the syntheticEmu monocot promoter (pEmuKN) was co-transferred with pU3Zald intosugarcane to provide a selectable marker. As a control, the ubi-luc andubi-gus reporter gene constructs pAHC18 and pU3ZGUS was co-transferredin the same way with pEmuKN into sugarcane. The stable transformedembryogenic callus was selected on medium containing geneticin andregenerated.

Crude protein extracts from leaves of a selection of Q63 plantsincluding untransformed controls, lines transformed with the gusreporter system, and lines selcted after co-bombardment with albD weretested for capacity to inactivate albicidin. TABLE 6 shows that noalbicidin detoxification was detected in negative control lines.However, activity was detected in various transformed lines, indicatingthat the albD gene had been stably integrated and expressed in thesetransgenic lines. Furthermore, the transgenic lines with albicidindetoxification activity as a result of expression of albD were resistantto leaf scald disease as indicated by absence of characteristic whitepencil lines on inoculated leaves (TABLE 6). Not all lines co-bombardedwith albD showed albicidin detoxification activity or diseaseresistance. This is the expected result because transgenic lines wereselected only for antibiotic G418 (geneticin) resistance, a phenotypeencoded by the aphA (nptl) gene. The efficiency of co-expression ofother genes co-bombarded with aphA under these condition is typically inthe range of 20% to 60%.

Challenging the Trangenic Plants with the Pathogen

After growth in the greenhouse for about 3 months, plants werechallenged with X. albilineans strain XA3 or XA15. FIGS. 12-13 show thatmany transgenic plants regenerated after co-bombardment with albD showedincreased resistance to leaf scald disease, as indicated by few or nocharacteristic white pencil line symptoms on inoculated leaves, whereascontrol lines not bombarded with albD typically developed multiple whitepencil lines. Absence of white pencil lines in transgenic lines wasassociated with capacity for detoxification of albicidin (TABLE 6).

CONCLUSION

A bacterial strain SB1403 producing a strong phytotoxin (albicidin)detoxification enzyme (AlbD enzyme) has been isolated and identified asErwinia herbicola (or Pantoea dispersa). SB1403 was very effective inthe biocontrol of sugarcane leaf scald disease caused by X. albilineans,and AlbD enzyme is one of the determinants of the biocontrol.

The gene encoding AlbD enzyme has been cloned and sequenced. The albDgene is a novel gene, there is not any significant sequence homology ateither DNA or protein levels to any other known DNA or proteinsequences.

The AlbD enzyme has been purified to homogeneity. The enzyme inactivatesalbicidin effectively at pH ranging from 5.8-8.0. The optimumtemperature for the enzyme activity is 28° C. The enzyme has no complexcofactor requirement.

An out of frame ATG (start) codon which would interfere with expressionin plant cells was removed by PCR. The PCR modified coding sequence ofalbD gene was cloned into a sugarcane expression vector including themaize ubiquitin promoter and nopaline synthase 3′ terminator. It hasbeen introduced into sugarcane by particle bombardment. The transgenicplants expressing AlbD enzyme were resistant to leaf scald diseasecaused by X. albilineans.

EXAMPLE 2 Biocontrol of Leaf Scald Disease in Sugarcane by a Strain ofPantoea dispersa which Inactivates Albicidin

MATERIALS AND METHODS

Bacteria, media and growth conditions. X. albilineans strain XA3 wasisolated from leaf scald diseased sugarcane as described previously(Birch and Patil, 1987, Physiological and Molecular Plant Pathology, 30,199-206). All other bacteria used in the study are listed in Table 7.Escherichia coli was grown in LM medium (Miller, 1972, Experiments inMolecular Genetics, Cold Spring Harbour, N.Y.: Cold Spring HarbourLaboratory Press) at 37° C. All other bacteria were grown in SP medium(Birch and Patil, 1985, J. Gen. Microbiol., 131, 1069-1075) at 28° C.Broth cultures were aerated by shaking at 180 rpm on an orbital shaker.

Preparation and assay of albicidin. Albicidins produced in culture by X.albilineans strain XA3 were purified as described previously (Birch andPatil, 1985, J. Gen. Microbiol., 131, 1069-1075; Birch et al., 1990, J.Gen. Microbiol., 136, 51-58). The mixture of albicidins obtained afterHW-40(S) chromatography was used in experiments reported here. Albicidinwas quantified as described previously (Birch and Patil, 1985, J. Gen.Microbiol., 131, 1069-1075), except that E. coli strain DH5 was used asthe indicator strain, and 1% agarose was used for overlayers.

Isolation of albicidin resistant bacteria. Leaf scald diseased sugarcanewas collected from disease resistance trials conducted by the Bureau ofSugar Experiment Stations in Brisbane, Australia. Leaf and stem samplesshowing symptoms of invasion by X. albilineans were surface sterilisedby 70% ethanol, then finely chopped, and suspended in sterilised waterwith shaking for 1 h before spreading over SP agar plates. Visiblydistinct colony types were restreaked to ensure purity of isolates,which were tested for albicidin resistance by streaking onto platescontaining specified concentrations of the antibiotic.

Test of albicidin resistance mechanism. Actively growing bacterialculture (optical density at 600 nm of 1.5) was added to an equal volumeof SP broth containing albicidin at a final concentration of 500 ngml⁻¹, then incubated at 28° C. for 6 h. Samples were placed on ice orboiled for 5 min, then centrifuged at 11020×g for 10 min. Supernatantswere assayed for albicidin, after exposure to UV (312 nm) for 10 min tokill cells in unboiled treatments.

Albicidin inactivation by cell free filtrates and cell extracts. StrainSB1403 was grown in broth for 40 h then chilled in ice and centrifugedat 11020×g for 10 min. The supernatant was filter sterilized (GelmanAcrodisc® 0.2 μm), then stored on ice or treated with 20 μg ml⁻¹protease K (signma) for 1 h at 28° C. Capacity to inactive albicidin inSP broth was then tested as described above.

To prepare cell extracts, cells from a 24 h broth culture were harvestedby centrifugation at 11020×g for 10 min, washed and resuspended in TEMMbuffer (10 mmol 1⁻¹ Tris pH 7.45, 10 mmol 1⁻¹ EDTA, 10 MMOL 1⁻¹ MgCl₂and 2 mmol 1⁻¹ β-mercaptoethanol), and disrupted by sonification on icewith a microprobe (Branson model 250), at 50% duty cycle and an outputof 25-45 W for 3 min of 8 s sonification followed by 8 s rest periods.Cell disruption was confirmed by phase contrast microscopy. Cell debriswas removed by centrifugation at 11020×g for 20 min at 4° C. Proteinconcentrations in cell extracts were measured by dye-binding (Bradford,1976, Analytical Biochemistry, 72, 248-254), using bovine serum albuminfor calibration.

Taxonomic identification and antibiosis assays. Strain SB1403 was testedfor oxidation and fermentation of glucose using the Hugh and Leifsontest (Collins and Lyne, 1984, Microbiological Methods, 5th edn., London:Butterworths), production of β-galactosidase (ONPG test), utilisation ofcarbon sources in the BIOLOG GN Microplate system (Biolog Inc.), andsolubility in 3% KOH as a predication of the Gram reaction (Suslow etal., 1982, Phytopathology, 72, 917-918).

Antibiotic production by strain SB1403 was tested using E. coli DH5a andX. albilineans XA3 as indicator strains. Cell free culture filtratesafter 1, 2 and 3 d growth in SP broth were tested using overlayer assaysas described for albicidin. Colonies after incubation for 2 d on SP agarwere killed by exposure to CHCl₃ vapour, then overlaid with theindicator bacteria.

Plant material and bacterial inoculation. Sugarcane variety Q44 which ishighly susceptible to leaf scald disease was used in all biocontrolexperiments. Single-node cuttings from healthy plants were grown in agreenhouse for about two months before inoculation with X. albilineansand P. dispersa strains by a decapitation method as described previously(Birch and Patil, 1983, Phytopathology, 73, 1368-1374). The inoculumconsisted of bacteria from actively growing cultures (2 d culture of X.albilineans and 24 h culture of P. dispersa), which were centrifuged for1 min at 11020×g, resuspended and diluted with sterilised water tospecified concentrations, and kept on ice until inoculation. Theinoculum was applied by coating onto the freshly cut surface of theplant.

Plants were inspected 2 weeks after inoculation for symptoms on cutleaves, and systemic symptom development was monitored for 6 months.Resolution of bacteria was attempted from inoculated leaves after 1month, and from young stem tissue 6 months after inoculation. SP platescontaining 200 μg ml⁻¹ ampicillin or 500 ng ml⁻¹ ampicillin or 500 ngml⁻¹ albicidin were used to selectively reisolate X. albilineans ZA3 andP. dispersa SB1403 respectively.

RESULTS AND DISCUSSION

Isolation of albicidin resistant bacteria. Of fifteen bacteria withdistinct colony characteristics isolated from X. albilineans infectedsugarcane, thirteen isolates proved resistant to albicidin at 50 ng ml⁻¹(the minimum inhibitory concentration for E. coli). Three isolates wereresistant to 1000 ng ml⁻¹ albicidin (TABLE 1).

Screening for bacteria that detoxify albicidin. Many bacteria activelyaccumulate albicidin from the surrounding medium. In the case of E. colithis process is known to involve active uptake via the Tsx outermembrane pore, and diminished uptake results in albicidin resistance(Birch et al., 1990, J. Gen. Microbiol., 136, 51-58). Some bacteria areresistant to albicidin due to production of an intracellular proteinwhich binds the antibiotic (Walker et al., 1988, Molecular Microbiology,2, 443-454; Basnayake and Birch, 1995, Microbiology, 141, 551-560).Albicidin is heat stable and little activity is lost at 100° C. for 30min, whereas bacterial cell membranes and many proteins are denatured byboiling, releasing reversibly bound albicidin. This allows a simpleassay to distinguish various mechanisms of albicidin resistance.Bacterial samples are mixed with albicidin solution, aliquots areremoved after incubation and kept on ice or boiled before assaying foralbicidin activity in the supernatants. When fresh bacterial culturesare used, the assay distinguishes toxin inactivation from other knownresistance mechanisms. Use of dense cell suspensions, cell extracts orculture supernatants in the assay further distinguishes toxin exclusion,toxin binding, probable resistant target, and intracellular versusexported proteins as resistance mechanisms.

Results using fresh cultures of the albicidin resistant bacteria fromsugarcane, compared to controls with known resistance mechanisms,indicate diverse resistance mechanisms including the first examples ofalbicidin detoxification (TABLE 7). Strain SB1403, which showed thestrongest albicidin detoxification, was characterized in more detail.Culture filtrates from this strain showed increasing extracellularactivity as cultures aged from 24 h to 40 h. Culture filtrate from a 40h culture abolished antibiotic activity of a 300 ng ml⁻¹ albicidinsolution during a 6 h incubation, and no antibiotic activity wasrecovered upon boiling the mixture. The capacity of culture filtrate toinactivate albicidin was abolished by treatment with protease K. Cellextracts of strain SB1403 also caused progressive and irreversibleinactivation of albicidin (FIG. 13). These observations indicateenzymatic detoxification of albicidin by strain SB1403.

Taxonomic identification. Strain SB1403 is a gram-negative, rod-shapedbacterium (0.6-1.0 μm×1.3-3.0 μm) with 4-8 peritrichous flagella.Colonies on SP agar are yellow. The strain was positive for oxidationand fermentation of glucose, production of β-galactosidase, metabolismof L-arabinose, cellobiose, glycerol, myo-inositol, maltose,N-acetyl-D-glucosamine, D-mannitol, D-mannose, L-rhamnose, sucrose andtrehalose. The strain was negative in tests for phenylalanine deaminase,arginine dihydrolase, ornithine decarboxylase, metabolism of malonate,D-adonitol, lactose, lactulose, I-erythritol, L-fucose, turanose,xylitol, D-galacturonic acid lactone and N-acetyl-D-galactosamine. Thesecharacteristics allow identification of strain SB1403 as Pantoeadispersa (Gavini et al., 1989, International Journal of SystematicBacteriology, 39, 337-345). However, strain SB1403 differed frompreviously characterized strains of P. dispersa by producing acid fromraffinose, sorbitol and α-methyl-D-glucoside.

Biocontrol of leaf scald by P. dispersa SB1403. P. dispersa was veryeffective as a biocontrol against X. albilineans when applied to woundedsugarcane at the same time as the pathogen (Table 8). Sugarcane varietyQ44 is highly susceptible to leaf scald disease, and develops severesymptoms in emerging leaves following inoculation with X. albilineans.The inoculum concentration used in this experiment resulted in death of50% of inoculated leaves within 2 weeks, and an average of 14characteristic white pencil lines power plant in surviving leaves. Inplants co-inoculated with P. dispersa SB1403, no leaves died and therewas a 98% reduction in the frequency of white pencil lines, even with aten-fold excess of X. albilineans cells in the inoculum.

Biocontrol agent P. dispersa SB1403 invaded wounded sugarcane leaveswithout causing any visible symptoms or any apparent adverse effect ongrowth of the inoculated sugarcane plants. Six months after inoculation,it was present in young stem tissue of apparently healthy sugarcaneplants at populations ca 10³-fold higher than similar organisms inwater-inoculated controls.

X. albilineans was readily reisolated from inoculated leaves showingwhite pencil lines. Six months after inoculation, over 90% of plantsinoculated with X. albilineans alone were dead. In contrast, plantsco-inoculated with the biocontrol agent developed no systemic leaf scaldsymptoms and the pathogen could not be reisolated, even on SP mediumcontaining ampicillin, which permits growth of the pathogen but not thebiocontrol agent.

Because X. albilineans is spread very efficiently during mechanicalharvesting of sugarcane (Taylor et al., 1988, Sugar Cane, 1988 (4),11-14), the high level of biocontrol provided by simultaneousapplication of P. dispersa SB1403 may be useful to restrict spread ofthe pathogen in the field. For example, the biocontrol agent could beapplied by dripping onto base cutters or through spray nozzles directedat the freshly cut stubble. A similar approach could be used inmechanical cutter-planters, which are typically already equipped with aspray or dip system to apply fungicide to the cut ends of stalk sectionsbefore planting.

Strains of Erwinia herbicola (syn. Pantoea spp.; Gavini et al., 1989,International Journal of Systematic Bacteriology, 39, 337-345) providingbiocontrol against fire blight caused by Erwinia amylovora (Vanneste etal., 1992, Journal of Bacteriology, 174, 2785-2796) or blackleg causedby Leptosphaeria maculans (Chakraborty et al., 1994, Letters in AppliedMicrobiology, 18, 74-76), produce antimicrobial substances antagonisticto those pathogens. P. dispersa SB1403 did not produce any detectableantibiotic effective against E. coli or X. albilineans. Rather, P.dispersa SB1403 produces a detoxification enzyme which protects thisbiocontrol agent against albicidin antibiotics produced by the pathogen.As albicidins are known to play a role in pathogenicity of X.albilineans, albicidin detoxification may act not only to favourcolonisation by P. dispersa in competition with X. albilineans at woundswhich are the primary sites of invasion, but also to protect the plantby removal of albicidin phytotoxins necessary as pathogenicity factorsfor establishment of the pathogen and development of systemic leaf scalddisease in sugarcane.

EXAMPLE 3 Further Studies

Experiments demonstrating the effect of expression of albicidindetoxification enzyme in X. albilineans. A 806 bp SphI-pVUII fragment,containing the intact albD gene (including promoter, structure gene andterminator region) of E. herbicola SB1403 was blunt-ended and ligatedinto the HindII site of suicide vector pJP5603. The ligation product wasused to transform E. coli JM109 (λpir). A recombinant clone, pJPAIdSPwas identified by restriction enzyme digestion and agrose gelelectrophoresis. It was transferred into the mobilising strain S17-1(λpir), and mobilised into X. albilineans XA3 which is resistant toampicillin. Exconjugant colonies were selected on the SP agar mediumcontaining 50 μg/mL kanamycin and 200 μg/mL ampicillin, and tested foralbicidin production and synthesis of AlbD enzyme. Genetically modifiedX. albilineans expressing albD failed to induce disease on susceptiblesugarcane, while the parent X. albilineans strain induced severesymptoms. This further supports the importance of albicidin inpathogenicity and the use of the albD gene to confer disease resistance.

Experiments further demonstrating resistance of various sugarcane linesto leaf scald disease. The albD gene was modified for expression inplants, and introduced into leaf scald susceptible sugarcane cultivarQ63 by particle bombardment. More than 60 lines cobombarded with albDand the NPT-II gene have been regenerated, of which approximately halfexpress the toxin resistance gene. Plants from 34 lines cobombarded withalbD and 20 control lines were challenged with X. albilineans. Tebtransgenic lines showed no disease symptoms under conditions whichcaused severe symptoms on controls (90% infection rate, average of 13white pencil lines per plant on inoculated leaves and rapid death ofmany plants). This confirms the effectiveness of albD as a leaf scalddisease resistance gene in transgenic sugarcane.

Experiments with the albicidin detoxification enzyme. The AlbD enzymeappears to be a hydrolyase. Two pieces of evidence support thissuggestion. Firstly, purified AlbD enzyme combined with purifiedalbicidin in water was found in inactivate albicidin. As no co-factorswere required and other known mechanisms of activation cannot operateunder these conditions, the mode of inactivation points to hydrolysis.Secondly, the inactivation reaction as analysed by HPLC showed the lossof albicidin with the production of two peaks representing the productsof the reaction. Again, this result points towards an hydrolysisreaction.

TABLE 1 Bacterial strains and plasmids Reference or Strain or Plasmid*Genotype or feature source X. albilineans XA3 Sugarcane leaf scaldpathogenic isolate, albicidin This work producer E. herbicola SB1403Alb^(r) This work E. herbicola Rifampincin resistant derivative ofSB1403 This work SB1403rif Alb^(r), Rif^(r) E. coli DH5α F⁻deoR endA1gryA96 hsdR17 (r⁻⁻ m⁺) Sambrook et al. Δ(lacZYA-argF)U169(φ80d lacZΔM15)recA1 (1989) relA1 supE44 thi-1λ⁻ Alb^(s) PLASMID pLAFR3 Cosmid vector,Tc^(r) Murphy, P. PBS SK+ Cloning and Sequencing vector, Ap^(r)Stratagene pKK223-3 Bacterial expression vector, Ap^(r) PharmaciapGEM-2T GST gene fusion vector, Ap^(r) Pharmacia pJP5603 Bacterialsuicide vector, Kn^(r) Penfold and Pemberton pSB6 albB gene from A.denitrificans cloned in Basnayake and pBluescriptll SK+, Ap^(r, Alb)^(r) Birch, 1995 pGEM-4Z DNA cloning vector, Ap^(r) Promega pBl101Clontech pEmuKN nptll gene fused to pEmu Monocots promoter, Ap^(r) Lastet al., 1991 pAHC18 luc gene fused to maize ubiquitin promoter, Ap^(r)Bruce et al., 1989 pU3Z Sugarcane expression vector containing This workubi∫intron promoter region and nos 3′ terminator sequence, Ap^(r)pU3Zald albD gene fused to ubi∫intron in vector pU3Z, Ap^(r) This workpU3ZGUS gus gene fused to ubi∫intron in vector pU3Z, Ap^(r) This workpTacAld albD gene fused to pTac promoter in vector This work pKK223-3,Ap^(r), Alb^(r) pGSTALD albD gene fused to GST gene in vector pGEM- Thiswork 2T, Ap^(r), Alb^(r) pJPAldHS An internal fragment of albD genecloned in This work suicide vector pJP5603, Ap^(r), Alb^(r)

TABLE 2 Bacteria isolated from X. albilineans infected sugarcane plantsRESISTANCE TO ALBICIDIN SUGARCANE (units/ml) ISOLATE ORIGIN 50 500 1000SB101 tip of inoculated leaf + + + SB107 tip of inoculated leaf + + +SB109 tip of inoculated leaf + − − SB501 middle section of inoculatedleaf + + − SB902 middle of natural infected leaf + − − SB903 middle ofnatural infected leaf + − − SB904 middle of natural infected leaf + − −SB905 middle of natural infected leaf + − − SB1301 top part of cane + +− SB1401 base part of cane + + − SB1402 base part of cane + + − SB1403base part of cane + + + SB1404 base part of cane + + − SB1405 base partof cane − − − SB1406 base part of cane − − −

TABLE 3 Albicidin resistant bacteria from sugarcane and their possibleresistance mechanism % ADDED ALBICIDIN ACTIVITY RECOVERED AFTERREACTION* PUTATIVE TREATMENT UNBOILED BOILED MECHANISM Alb^(r)(controls) 100 90 +A. denitrificans 59 68 toxin binding +DH5α(pSB6) 0 55toxin binding +RR1Alb^(r) 95 94 toxin exclusion +SB1401 0 23 toxinbinding +SB1402 36 64 +SB501 91 73 toxin or exclusion? +SB1301 86 73 or+SB1404 91 75 resistance target? +SB101 45 41 toxin inactivation +SB10746 41 +SB1403 9 0

TABLE 4 Effect of E. herbicola SB1403 in biocontrol of leaf scalddisease of sugarcane* X.a₁₀ X.a₁₀ X.a₁₀ Inoculum Water X.a₁₀ E.h₁ E.h₁₀E.h₁₀₀ E.h₁₀ Dead leaves 0  20 0 0 0 0 White pencil lines 0 139 2 3 0 0

TABLE 5 Biocontrol effect of E. herbicola SB1403rif, SM1, SM18 on leafscald disease of sugarcane caused by X. albilineans XA3 No. of whiteTREATMENT pencil lines No. of dead leaves Water 0 0 XA3(10) 129 6XA3(10), SB1403rif(10⁻¹) 1 0 XA3(10), SB1403rif(10⁻²) 3 0 XA3(10),SB1403rif(10⁻³) 5 0 XA3(10), SM1(10⁻¹) 8 0 XA3(10), SM1(10⁻²) 9 0XA3(10), SM1(10⁻³) 26 0 XA3(10), SM18(10⁻¹) 9 0 XA3(10), SM18(10⁻¹) 11 0

TABLE 6 Correlation between albicidin detoxification and leaf scaldresistance as indicated by absence of white pencil line symptoms incontrol and transgenic lines of sugarcane variety Q63 AlbD activity(zone White pencil lines per reduction in mm Plant/Line plant diameter)untransformed 1 6.3 0 untransformed 1 8.1 0 gus-1 5.8 0 gus-2 6 0 albD804-3b 0 8 albD 804-3c 0 7 albD 804-3d 0 9 albD 811-4d 0 7 albD 811-4f 09 albD 811-4k 6.7 0 albD 811-5a 0 8 albD 811-5b 0 9 albD 811-5c 8.3 0albD 811-6a 0 9 albD 825-2a 0 9 albD 825-2c 0 8

TABLE 7 Albicidin resistant bacteria from sugarcane, and possibleresistance mechanisms Level of albicidin Albicidin recovered Probableresistance after reaction (%) Resistance Bacterial strain* (ng/ml⁻¹)Unboiled Boiled Mechanism E. coli RR1 <50 78 87 none E. coli RR1Alb^(r) >1000 95 94 exclusion E. coli DH5 pSB6 >1000 0 55 binding A.denitrificans SO9 >1000 59 68 binding SB501 500 91 73 exclusion orresistant target SB1301 500 86 73 SB1404 500 91 75 SB1401 500 0 23binding resistant target SB1402 500 36 64 SB101 >1000 45 41detoxification SB107 >1000 46 41 SB1403 >1000 9 0

TABLE 8 Biocontrol of sugarcane leaf scald disease by P. dispersaSB1403* INOCULUM† X. albilineans P. dispersa WHITE PENCIL XA3 SB1403DEAD LEAVES LINES 0 0 0 6 10 0 20 139 10 1 0 2 10 10 0 3 10 100 0 0 0 100 0

LEGENDS

TABLE 1

* Plasmids constructed in albD gene cloning and sequencing are shown inFIG. 2.

TABLE 3

* The final concentration in the reaction mixture was 500 μ/mL, and eachtreatment has three replicates.

TABLE 4

* Each treatment has 10 plants. Symptoms were recorded from 4 newlyemerged leaves after inoculation. X.a=X. albilineans XA3,E.h=E.herbicola SB1403. Numbers next to X.a and E.h represent relativeproportion of bacterial cell numbers, with 10=4×10⁸ C.F.U. (colonyforming unit). The inoculation volumn for each plant was 200 μL.

TABLE 5

* Each treatment has 6 plants. Symptoms were recorded from 4 newlyemerged leaves after inoculation. Numbers in bracket represent relativeproportion of bacterial cell numbers used in inoculation, with 10=4×10⁸C.F.U. The inoculum volume for each plant was 200 μL.

TABLE 7

* Strains in the top panel are controls with known resistance mechanisms(Birch et al., 1990; Basnayake and Birch, 1995). Strains commencing withSB were isolated from diseased sugarcane in this study. Results aremeans of three replicates.

TABLE 8

* Symptoms were recorded 14 d after inoculation of sugarcane varietyQ44, which is highly susceptible to leaf scald disease. Results aretotals from four inoculated leaves on ten plants per treatment.

† Inoculum consisted of 200 μL volume containing the pathogen andbiocontrol agent at the ratios shown, where 10 equals 4×10⁸ colonyforming units.

FIG. 1

Time course of albicidin detoxification by cell free extracts of E.herbicola SB1403. Denatured (boiling 5 min) and undenatured cell freeextracts containing 100 μg total protein were added to TEMM buffercontaining 300 ng of albicidin in a final volume of 100 μL, andincubated at 28° C. The reaction was stopped by boiling for 3 min. Thereaction mixture was 10 centrifuged (14000 rpm) for 5 min beforebioassay.

FIG. 2

Physical map of pQZB103 and its derivatives. The cloning vectorpBluescriptII SK+ is represented by open boxes at both ends of thelinearlized plasmid map. Except for pQZB103 and pQZH301, all otherplasmids were generated by ExoIII unidirectional deletion. Albicidinresistance or sensitivity of each plasmid encoded in E. coli DH5α isindicated.

FIG. 3

Nucleotide sequence of the albD gene (SEQ ID NO:2) (FIGS. 3B-3F) and thepredicated amino acid sequence (SEQ ID NO:1) (FIG. 3A) of its geneproduct. The PCR primers (SEQ ID NO:7 and SEQ ID NO:8) used foramplification of the coding region and elimination of a spurious (ATG)start codon are also shown.

FIG. 4

Best match of amino acids sequence of albD gene product (A) (SEQ IDNO:4) to the first 16 amino acids in the N-terminal of albicidin bindingproteins encoded by A. denitrificans albB gene (B) (SEQ ID NO:5) and byK. oxytoca albA gene (C) (SEQ ID NO:6) Symbols: Double dot, identicalamino acids; single dot, amino acids with similar properties in theirside chains.

FIG. 5

Construction of suicide plasmid clone pJPAldHS for site-directedmutagenesis of albD gene in E. herbicola SB1403. (A) The internalHincII-StuI fragment (Shown as shadowed AldHS fragment, 326 bp) of albDgene was isolated and its relative distances to the ATG initiation andTAG stop codons were indicated. (B) Map of pJPAldHS. The AldHS fragmentwas ligated to the HincII site of suicide vector pJP5603, and theorientation of the AldHS fragment in pJPAldHS was determined by HincIIand BamHI restriction enzymes double digestion.

FIG. 6

Inactivation of albicidin by E. coli DH5α [pQZE533] and E. coli DH5α[pSB6]. Plasmids pQZE533 and pSB6 contain albD gene cloned from E.herbicola SB1403 and albB gene from A. denitrificans, respectively.

FIG. 7

Construction of GST-albD gene fusion plasmid for purification of AlbDenzyme protein. (A) A 790 bp albD structural gene fragment was amplifiedby PCR using a pair of oligonucleotides primers from the plasmid clonepQZE533 which contains the intact albD gene from E. herbicola SB1403.The 5′ primer (SEQ ID NO:7) covers the ATG initiation codon (underlined)region of albD gene, and contains a mismatch nucleoside C. (indicated by*) to replace the original nucleoside G. The 3′ primer (SEQ ID NO:8)spans the region 38 bp downstream of the TAG termination codon. TheBamHI and PvuII restriction enzyme sites on the oligonucleotides primersare indicated. (B) The PCR amplified albD structural gene fragment wasdigested by BamHI and PvuII and ligated to the GST gene fusion vectorPGEX-2T linealized by BamHI and SmaI. The sequences (SEQ ID NO:10) inthe fusion region are shown and the cleavage-recognition sequence of thesite-specific protease Thrombin is indicated (SEQ ID NO:9). (C) Map ofGST-albD gene fusion construct pGSTALD.

FIG. 8

Effect of temperature on AlbD enzyme activity. Purified AlbD enzyme andalbicidin were mixed in 0.2M phosphate buffer, pH7.0 in finalconcentrations of 2 ng/μL AlbD enzyme and 15 u/μL albicidin; thereaction mixtures were incubated in water baths preset in differenttemperatures for 30 min. The reaction was stopped by boiling for 3 min.The albicidin remaining in the reaction mixture was assayed. Symbol:open bar, albicidin+AlbD; striped bar, albicidin only blank control. Thealbicidin used in this experiment was further purified using DE52chromatography.

FIG. 9

Effect of pH on AlbD enzyme activity, the condition for the enzyme assaywere described in FIG. 8 legend except the albicidin solutions wereprepared in phosphate buffers of different pH. Symbol: solid bar,albicidin+AlbD; open bar albicidin only blank control.

FIG. 10

(A) The PCR amplified coding sequence of albD gene was fused to tacpromoter for expressing of AlbD enzyme in E. coli. A pair of PCR primersflanking the start codon (underlined) (SEQ ID NO:7) and terminationregion (38 bp downstream the TAG stop codon) (SEQ ID NO:8) was used toamplify albD structural gene portion using plasmid clone pQZE533 astemplate. The 790 bp PCR product was digested by BamHI and PvuII, bluntended by Klenow DNA polymerase before ligated to SmaI site of bacterialexpression vector pKK223-3. (B) Map of resultant plasmid clone pTacAldshowing right orientated albD structural gene portion is under thecontrol of promoter Ptac.

FIG. 11

(A) Map of sugarcane expression vector pU3Z. Construction approacheshave been described in Materials and Methods section. (B) The codingsequence of albD was amplified from plasmid clone pQZE533. The PCRproduct was digested with BamHI and PvuII and ligated to BamHI and SmaIlinearlized vector pU3Z. (C) Map of resultant construct pU3ZaId.

FIG. 12

Frequency distribution of disease severity in sugarcane cultivar Q63plant lines regenerated from callus co-bombarded with albD (2-10replicate plants per line from 19 lines), and control lines notbombarded with albD (2-6 replicate plants per line from 9 lines).Experiment A, inoculated with X. albilineans XA3.

FIG. 13

Frequency distribution of disease severity in sugarcane cultivar Q63plant lines regenerated from callus co-bombarded with albD (2-10replicate plants per line from 34 lines), and control lines notbombarded with albD (2-6 replicate plants per line from 20 lines).Experiment B, inoculated with X. albilineans XA15.

12 1 235 PRT Erwinia herbicola 1 Met Asp Lys Ser Asp Leu Thr Glu Thr SerArg Ile Lys His Gly Glu 1 5 10 15 Glu Ala Phe Asp Val Thr Leu Leu GlnVal Lys Gly Ala Thr Arg Cys 20 25 30 Ile Leu Phe Ala Ala Gly Leu Ser GlySer Pro Leu Arg His Leu Glu 35 40 45 Leu Leu Gln Thr Phe Ala Arg His GlyVal Ser Val Val Ala Pro His 50 55 60 Phe Glu Arg Leu Thr Ser Pro Val ProThr Arg Ala Glu Leu Leu Glu 65 70 75 80 Arg Cys Gln Arg Leu Ala Arg AlaGln Asn Glu Phe Cys Ser Gly Tyr 85 90 95 Ala Ser Val Thr Gly Val Gly HisSer Leu Gly Ser Val Ile Leu Leu 100 105 110 Leu Asn Ala Gly Ala Ile AlaMet Thr Ser Ala Gly Glu Ser Val Val 115 120 125 Phe Ala Gly Asp Arg MetLeu His Arg Leu Ile Leu Leu Ala Pro Pro 130 135 140 Ala Asp Phe Phe GlnAla Pro Ser Ala Leu Ala Ala Val Asn Val Pro 145 150 155 160 Val His IleTrp Ala Gly Glu Lys Asp Ser Leu Thr Pro Pro Ser Gln 165 170 175 Ala CysPhe Leu Lys Gln Ala Leu Glu Gly Tyr Thr Gln Thr Tyr Leu 180 185 190 CysVal Met Glu Glu Ala Gly His Phe Thr Phe Met Asn Thr Leu Pro 195 200 205Pro Gln Val Thr Asp Ser His Pro Ser Arg Glu Ala Phe Leu Leu Asp 210 215220 Leu Gly Glu Asn Ile Ala Arg Leu Val Thr Asp 225 230 235 2 1016 DNAErwinia herbicola 2 atgcagaggg gctcaatgac gtttcatccc aatgtcctgaccagtcataa ttcaccaagc 60 cgaggtttgc tgtgtggcag aatggcatcc aacgcgtaaaggtggcgaga gcctgttaat 120 atttttgaca atcggttaag cgggatgcgt tttgatggacaaaagtgatc tcacggaaac 180 gtctcggatc aaacatgggg aagaggcgtt tgacgtcaccttattgcagg ttaagggggc 240 gacgcgctgt atcctttttg ctgcggggct gagcggcagtccgctgcgcc atcttgaact 300 tctccagacc tttgcccgcc atggcgtttc cgttgtcgcgccacactttg aacggttgac 360 ctcacccgtg cccaccagag ctgaattact ggaacgctgccagcggcttg cgcgggctca 420 gaatgaattt tgtagcggtt atgcgtcggt taccggtgttggccactccc tgggtagcgt 480 gattttattg ctgaatgccg gggctatagc gatgacaagcgcaggggaat cggttgtttt 540 cgccggcgac cggatgttgc atcgacttat tttactggcaccgcccgccg attttttcca 600 ggctccgtct gcgctggcag cggtgaacgt acctgttcacatctgggcag gtgaaaagga 660 cagcctgacg cccccgtccc aggcctgctt tcttaaacaggcactggagg gttacacgca 720 gacttatctc tgtgtgatgg aagaggccgg gcattttaccttcatgaata ccttgcctcc 780 gcaggtaacc gattcacatc cgtcgcggga ggcctttcttttagatttgg gcgaaaacat 840 agcccggctg gtgactgatt agcacagagg gcggggcgatgagatttttg cagggataac 900 ctcttccagc tgatacgatt caatcatact catcaaaagcatcatttcat cctgtcttag 960 gggctattgt gaaacagaaa tcggccctat agtgagtcgtattacgcccg ctcgaa 1016 3 704 DNA Erwinia herbicola 3 atggacaaaagtgatctcac ggaaacgtct cggatcaaac atggggaaga ggcgtttgac 60 gtcaccttattgcaggttaa gggggcgacg cgctgtatcc tttttgctgc ggggctgagc 120 ggcagtccgctgcgccatct tgaacttctc cagacctttg cccgccatgg cgtttccgtt 180 gtcgcgccacactttgaacg gttgacctca cccgtgccca ccagagctga attactggaa 240 cgctgccagcggcttgcgcg ggctcagaat gaattttgta gcggttatgc gtcggttacc 300 ggtgttggccactccctggg tagcgtgatt ttattgctga atgccggggc tatagcgatg 360 acaagcgcaggggaatcggt tgttttcgcc ggcgaccgga tgttgcatcg acttatttta 420 ctggcaccgcccgccgattt tttccaggct ccgtctgcgc tggcagcggt gaacgtacct 480 gttcacatctgggcaggtga aaaggacagc ctgacgcccc cgtcccaggc ctgctttctt 540 aaacaggcactggagggtta cacgcagact tatctctgtg tgatggaaga ggccgggcat 600 tttaccttcatgaatacctt gcctccgcag gtaaccgatt cacatccgtc gcgggaggcc 660 tttcttttagatttgggcga aaacatagcc cggctggtga ctga 704 4 16 PRT Erwinia herbicola 4Leu Phe Ala Ala Gly Leu Ser Gly Ser Pro Leu Arg His Leu Glu Leu 1 5 1015 5 16 PRT Alcaligenes dentrificans 5 Met Tyr Asp Lys Tyr Phe Ser ArgGlu Glu Leu Ala Arg Leu Pro Leu 1 5 10 15 6 16 PRT Klebsiella oxytoca 6Met Tyr Asp Arg Trp Phe Ser Gln Gln Glu Leu Gln Val Leu Pro Phe 1 5 1015 7 25 DNA Erwinia herbicola 7 ttaagcggga tccgttttga tggac 25 8 25 DNAErwinia herbicola 8 gattgaatcg tatcagctgg aagag 25 9 10 PRT Erwiniaherbicola 9 Leu Val Pro Arg Gly Ser Val Leu Met Asp 1 5 10 10 30 DNAErwinia herbicola 10 ctggttccgc gtggatccgt tttgatggac 30 11 14 RNAErwinia herbicola 11 aucaccuccu uucu 14 12 16 PRT Artificial SequenceDescription of Artificial Sequence Albicidin Binding Peptide Motif 12Met Tyr Xaa Xaa Xaa Phe Ser Xaa Xaa Xaa Leu Xaa Xaa Leu Xaa Leu 1 5 1015

I claim:
 1. An isolated nucleotide sequence encoding an albicidindetoxification enzyme comprising the sequence of amino acids set forthin SEQ ID NO:1.
 2. The isolated nucleotide sequence of claim 1 whereinthe nucleotide sequence comprises the sequence of nucleotides set forthin SEQ ID NO:2.
 3. The isolated nucleotide sequence of claim 1 whereinthe nucleotide sequence comprises the sequence of nucleotides set forthin SEQ ID NO:3.
 4. An isolated nucleotide sequence which hybridizesunder stringent conditions with the sequence of nucleotides set forth inSEQ ID NO:2 or SEQ ID NO:3.
 5. The isolated nucleotide sequence of claim4 wherein said nucleotide sequence is obtained from a bacterium.
 6. Theisolated nucleotide sequence of claim 4 wherein said nucleotide sequenceis obtained from a strain of Erwinia or Pantoea.
 7. A method ofproducing a transgenic plant with enhanced resistance to albicidin andleaf scald disease, said method comprising the steps of introducing andexpressing the nucleotide sequence of any one of claims 1 to 6 in aplant, plant part or plant cell, and growing the plant, plant part orplant cell to produce the transgenic plant.
 8. A method of producing atransgenic plant with enhanced resistance to albicidin and leaf scalddisease, said method comprising the steps of introducing and expressingthe nucleotide sequence of any one of claims 1 to 6 in a plant, plantpart or plant cell, and growing the plant, plant part or plant cell toproduce the transgenic plant, wherein the step of introducing thenucleotide sequence is effected by particle bombardment.
 9. A method ofproducing a transgenic plant with enhanced resistance to albicidin andleaf scald disease, said method comprising the steps of introducing andexpressing the nucleotide sequence of any one of claims 1 to 6 in aplant, plant part or plant cell, and growing the plant, plant part orplant cell to produce the transgenic plant, wherein said plant, plantpart or plant cell is or is obtained from a sugarcane variety.
 10. Amethod of producing a transgenic plant with enhanced resistance toalbicidin and leaf scald disease, said method comprising the steps ofintroducing into a plant, or plant part or cell thereof a vectorcomprising the nucleotide sequence of any one of claims 1 to 6 whereinsaid nucleotide sequence is operably linked to one or more regulatorynucleotide sequences, and growing said plant or plant part or cellthereof to produce said transgenic plant.
 11. A method of producing atransgenic plant with enhanced resistance to albicidin and leaf scalddisease, said method comprising the steps of introducing into a plant,or plant part or cell thereof a vector comprising the nucleotidesequence of any one of claims 1 to 6 wherein said nucleotide sequence isoperably linked to one or more regulatory nucleotide sequences, andgrowing said plant or plant part or cell thereof to produce saidtransgenic plant, wherein the step of introducing the vector is effectedby particle bombardment.
 12. A method of producing a transgenic plantwith enhanced resistance to albicidin and leaf scald disease, saidmethod comprising the steps of introducing into a plant, or plot part orcell thereof a vector comprising the nucleotide sequence of any one ofclaims 1 to 6 wherein said nucleotide sequence is operably linked to oneor more regulatory nucleotide sequences, and growing said plant or plantpart or cell thereof to produce said transgenic plant, wherein saidplant, plant part or plant cell is or is obtained from a sugarcanevariety.
 13. A transgenic plant with enhanced resistance to albicin andleaf scald disease, said plant comprising the nucleotide sequence of anyone of claims 1 to 6 wherein said sequence is operably linked to one ormore regulatory nucleotide sequences.
 14. A transgenic sugarcane plantwith enhanced resistance to albicidin and leaf scald disease, said plantcomprising the nucleotide sequence of any one of claims 1 to 6 whereinsaid sequence is operably linked to one or more regulatory nucleotidesequences.
 15. A transgenic plant with enhanced resistance to albicidinand leaf scald disease, said plant comprising the nucleotide sequence ofany one of claims 1 to 6 wherein said sequence is operably linked to oneor more regulatory nucleotide sequences and is stably incorporatedwithin the genome of cells of said plant.
 16. A transgenic sugarcaneplant with enhanced resistance to albicidin and leaf scald disease, saidplant comprising the nucleotide sequence of any one of claims 1 to 6wherein said sequence is operably linked to one or more regulatorynucleotide sequences and is stably incorporated within the genome ofcells of said plant.
 17. A vector comprising the nucleotide sequence ofany one of claims 1 to
 6. 18. A vector comprising the nucleotidesequence of any one of claims 1 to 6 wherein said nucleotide sequence isoperably linked to one or more regulatory nucleotide sequences.